Riassunto esame Genetica, Prof. Kater Martin, libro consigliato Genetica - un approccio molecolare, Peter J. Russell

Genetics: Mendel's Experiments

Mendel is considered to be the founder of genetics as he proposed the concept of gene in 1865. His prescient deduction of paired elements that break up their association and separate into different daughter cells is what makes him the founder of genetics. However, his work wasn’t recognized until about 1900, after his death, when three other botanists came to the same conclusions as Mendel.

Mendel's Background and Experiments

He was born in the district of Moravia, then part of the Austro-Hungarian Empire. In 1843, he was admitted to the Augustinian monastery of Bruenn (now Brno of the Czech Republic). In 1854, he started a series of experiments that involved the Pisum sativum plant in order to explain the mechanism of inheritance.

Mendel’s studies are a good example of the scientific method:

• Research material well suited to the problem at hand;
• Designed his experiments carefully;
• Collected large amounts of data;
• Used mathematical analysis to show that his results were consistent with his explanatory hypothesis;
• The predictions of the hypothesis were then tested in a new round of experimentation.

Reasons for Using Pisum Sativum

The reasons why Mendel decided to use Pisum sativum were:

• Peas were easily available in a wide array of distinct shapes and colors;
• Easy to self or cross-pollinate;
• Inexpensive;
• Little space required;
• Short generation time;
• Many seeds per plant.

In particular, Mendel precisely chose and studied seven different phenotypes, each of which is present in two different variants: flowers' petals' color (purple or white); seed's color (yellow or green), seed's shape (round or wrinkled); pod's color (green or yellow); pod's shape (inflated or pinched); stem's length (long or short); flowers' position (axial or terminal).

Mendel liked to work with seed color or shape phenotypes because in pea these phenotypes depend on the genetic constitution of the seed itself and not the maternal parent. This means that you can observe the next generation phenotype without having to grow the seeds.

Phenotype and Genotype

A phenotype is the set of observable characteristics or traits of an organism, while the genotype is the genetic constitution of an organism. A phenotype can be dominant (X), if it masks the other phenotype, or recessive (x).

Mendel's Methodology

Mendel’s method consisted in:

• Analysis of a single phenotypic character at the time;
• Use of pea plants that differ only for a single easily observable character;
• Creation of a pure line for each character. Pure lines always give offspring with the same character;
• Quantitative approach: count of the observed phenotypes and their proportions.

Experiments with Pure Lines

First, Mendel let 34 different variants of pea plants self-pollinate themselves for several generations in order to obtain pure lines of different variants. He then chose the seven different phenotypes.

The plants from a pure line form the parental generation (P), while the offspring of two parental plants form the first filial generation (F1), whose offspring is the second filial generation (F2) and so on.

The first experiment consisted in the monohybrid cross between two organisms of different pure lines: one had round seeds, while the other had wrinkled seeds. The result was an F1 with round seeds only.

Results and Inferences

Mendel then let the plants of F1 self-pollinate themselves. The result was an F2 with round or wrinkled seeds in the same pod. Therefore, the wrinkled seed is the recessive variant because it was masked in F1 peas, but it reappears in F2. Mendel counted the round seeds and the wrinkled ones and he obtained a ratio of 3:1.

To confirm his results, Mendel took 519 F2 round peas and grew plants from them and selfed them: 166 plants of them gave only round peas, while the other 353 plants gave a mixture of round and wrinkled, with the same ratio of 3:1. He also took F2 wrinkled peas, grew plants from them and selfed them: all the peas of the offspring were wrinkled.

Existence of Genes

Mendel explained these results as follows:

• The existence of genes: there are hereditary determinants of a particular nature. We now call these determinants “genes”;
• Genes are in pairs: alternative phenotypes of a character are determined by different forms of a single type of gene (alleles). In adult pea plants, each type of gene is presented twice in each cell (gene pair). Organisms that contain two copies of the same specific allele of a certain gene are called homozygous, while organisms that contain two different alleles of a certain gene are called heterozygous. The F1 peas are all heterozygous and have round seeds because the round variant is dominant over the wrinkled one;
• The principle of segregation: the members of the gene pairs segregate (separate) equally into the gametes (egg or sperm);
• Gametic content: consequently, each gamete carries only one member of each gene pair;
• Random fertilization: the union of one gamete from each parent to form the first cell (zygote) and so a new offspring is random. Gametes combine without regard to which member of a gene pair is carried.

The peas obtained from F1 seeds produce the same quantity of two types of gametes: those with the dominant allele and those with the recessive one. Therefore, three different types of genotypes are produced: XX, Xx, xx. As a consequence of the random fusion of the gametes, the relative proportion of these zygotes is 1:2:1.

Law of Segregation

Thus Mendel proposed the first law or law of segregation, which states that, during the production of gametes, the two copies of each hereditary factor segregate so that offspring acquire one factor from each parent.

Law of Independence

Mendel also conducted experiments that focused on two copies of alternative phenotypes at the same time by crossing dihybrid plants, which are heterozygous for two traits. The results were the same as the first experiments. By selfing the dihybrid plants of F1, two results were obtainable: if the alleles for the seed’s shape and color were inherited together, the phenotypic ratio would be 3:1 between round-yellow seeds and wrinkled-green seeds; if the alleles were inherited independently, the phenotypic ratio would be 9:3:3:1 between round-yellow seeds, round-green seeds, wrinkled-yellow seeds and wrinkled-green seeds.

Since Mendel obtained the latter result, he was able to propose the second law or law of independence, which states that the laws of chance govern how the chromosomes align during meiosis and which particular characteristics of the parental pairs will occur in each individual offspring.

The 9:3:3:1 ratio is deducted by considering the two traits independently: by crossing monohybrids of each trait, ¾ of the offspring express the dominant trait, while the other ¼ express the recessive one. Now, if the two traits are considered together:

• ¾ * ¾ = 9/16 of the total offspring express the two dominant traits (yellow round peas);
• ¾ * ¼ = 3/16 of the total offspring express one of the two dominant traits (yellow wrinkled peas);
• ¼ * ¾ = 3/16 of the total offspring express the other dominant trait (green round peas);
• ¼ * ¼ = 1/16 of the total offspring express the two recessive traits (green wrinkled peas).

Law of Dominance

The third law or law of dominance states that one factor in a pair of traits dominates the other in inheritance unless both factors in the pair are recessive.

The Hereditary Material is DNA

Mendel’s experiments indicate the existence of genes that control certain characters. Now the question was if these hereditable genes were formed by DNA or by proteins.

Discovery of DNA as Genetic Material

In 1869, Miescher discovered a new type of weak acidic chemical that is present in large quantities in nuclei of leucocytes and he suggested that this is the substance of which genes are made of. It’s now known that this chemical is deoxyribonucleic acid (DNA).

After 1870, the importance of the nuclei becomes evident. Using a microscope, it was observed that, during fertilization, the nuclei of sperm and egg cells fuse. The next step was the discovery of chromosomes that are present in the nuclei.

Around 1900, it was generally known that: the number of chromosomes are specific for species; their number is relatively constant in cells; they are separated equally during divisions. These observations supported the idea that these chromosomes are responsible for the heredity.

Around 1920, there was several indirect evidence that chromosomes contain DNA. Chromatin in cells is composed roughly of 50% nucleic acids and 50% proteins. The scientific establishment believed that the genetic material contained proteins because they are macromolecules with known functions and are highly variable in structure. Others instead believed that the genetic material is composed of DNA because it’s a highly stable molecule, its amount is the same in all cells and gametes have half the amount of somatic cells.

The scientists that wanted to demonstrate the function of DNA had two problems: they had not only to show that DNA is the genetic material, but they also had to demonstrate that proteins do not have this function.

Key Experiments Demonstrating DNA is Genetic Material

The following experiments demonstrated that DNA is the hereditary material:

1. Griffith, 1928
   Griffith studied the bacterium Streptococcus pneumoniae, which causes pneumonia. In particular, he studied two different strains: S (“smooth”), which consists of smooth colonies because of the presence of a polysaccharide capsule and it’s virulent; R (“rough”), which consists of rough colonies because of the absence of the polysaccharide capsule and it isn’t virulent. There are several variants of S strain, each of which is characterized by a different capsule’s composition. Griffith studied IIS and IIIS strains. Griffith injected different strains into some mice: those which were infected with IIR bacteria (which derived from a mutated IIS bacteria) survived, while those which were infected with a IIIS bacteria died. However, if IIIS bacteria were killed by heat before the injection, the mice survived. Then, Griffith injected a mixture of alive IIR bacteria and dead IIIS bacteria into the mice. The result was that all mice died and IIIS bacteria were found in their blood. Griffith suggested that some IIR bacteria transformed into smooth and virulent bacteria due to the interaction with dead IIIS bacteria. Griffith called the agent responsible for the exchange of the genetic material “transforming principle”;
2. Avery, MacLeod, and McCarty, 1944
   They tried to identify the “transforming principle” discovered by Griffith by studying the transformation of R bacteria into S bacteria. They subjected IIIS bacteria to lysis and separated the cell extract from the cell debris by centrifugation. Then, they incubated the extract with alive IIR bacteria and grew them on a medium. IIIS bacteria appeared on the medium, thus demonstrating that the extract contained the “transforming principle”, which derived from the IIIS bacteria and was able to transform IIR bacteria into IIIS bacteria. One of the components of the extract (polysaccharides, proteins, DNA or RNA) must have been the “transforming principle”. They subjected the cell extract to enzymatic treatments in order to degrade its different components one at a time. They observed that the transformation didn’t take place after the degradation of DNA only, meaning that DNA must be the “transforming principle”;
3. Hershey and Chase, 1952
   They confirmed the results obtained by Avery by studying the T2 phage, which is a virus that infects the bacterium Escherichia coli by injecting its genetic material into the bacterium, while its outer envelope (phage ghost) stays outside the cell. They knew that T2 is composed of DNA and proteins only, but they didn’t know which of them formed its genetic material and was injected into the bacteria. They grew colonies of E. coli on a medium containing P or S. The reason why they used these isotopes was that DNA contains P and not S, while proteins contain S and not P. Then, they injected the virus into all the cultures and gathered the phage progeny, which incorporated the marked DNA or the marked proteins. Then, they injected viruses with marked DNA into a culture of E. coli and viruses with marked proteins into another culture. In the former case, most of the isotopes (P) were found inside the bacteria; in the latter case, most of the isotopes (S) were found inside the phage ghosts.

DNA's Structure

DNA and RNA are polymers composed of monomers called nucleotides. Each nucleotide contains a pentose sugar (deoxyribose in DNA, ribose in RNA), a nitrogen base (molecule containing N) and a phosphate group.

Nitrogen bases are divided into two classes: pyrimidines (cytosine, thymine, uracil), which consist of a single ring of six atoms, and purines (adenine, guanine), which consist of two condensed rings of five and six atoms. DNA contains C, T, A and G while RNA contains U instead of T.

The nitrogen base forms a glycosidic bond with the pentose sugar’s C1, thus forming a nucleoside. This bond involves the N in position 1 in pyrimidines or in position 9 in purines. When the phosphate group is added to the pentose sugar’s C5 by forming a phosphodiester bond, a nucleotide is formed.

Polynucleotide Formation

DNA contains four types of nucleotides: deoxyadenosine 5’-monophosphate (deoxyadenilate or dAMP), deoxyguanosine 5’-monophosphate (deoxyguanylate or dGMP), deoxycytidine 5’-monophosphate (deoxycytidylate or dCMP) and deoxythymidine 5’-monophosphate (deoxythymidylate or dTMP).

To form polynucleotides, the phosphate group attached to the C5 of each nucleotide forms a phosphodiester bond with the hydroxyl group attached to the C3 of another nucleotide. Thus, one extremity ends with a hydroxyl group attached to the C3 of the last nucleotide, while the other extremity ends with a phosphate group attached to the C5 of the last nucleotide.

Discovery of DNA Structure

DNA’s structure was discovered in 1953 by Watson and Crick, who worked from two kinds of clues made before:

• Chargaff’s Rules, 1950
  Chargaff analyzed a large section of DNA from different organisms and established empirical rules about the amount of each component of DNA: 50% of nitrogen bases consists of purine and the other 50% consists of pyrimidines; the amount of A is equal to the amount of T and the amount of G is equal to the amount of C; the total amount of purines (A+G) is equal to the total amount of pyrimidines (C+T); the ratio A/T and G/C is always 1, while the ratio (A+T)/(G+C) varies;
• X-ray Experiments of Franklin and Wilkins, 1951
  This technique uses an X-ray beam directed towards the molecules and it’s diffracted by the molecules’ atoms in a way that depends on the atomic weight and the spatial organization of the molecules. They revealed that DNA is long and skinny, it has two similar parts that are parallel to each other and run along the length of the molecule and it’s helical.

According to Watson and Crick’s model, DNA has the following characteristics:

• It’s composed of two polynucleotide chains which coil around each other to form a double right-handed helix, meaning that the chains coil around each other clockwise;
• The chains are antiparallel, so they run in opposite directions (one from 3’ to 5’, the other from 5’ to 3’);
• The backbone of the DNA strand is made from alternating phosphate and sugar groups that face outwards, while the nitrogen bases of both chains face inwards and are stacked one above the other;
• Nitrogen bases are held together by hydrogen bonds in a complementary way: A only bonds with T (two hydrogen bonds), while G only bonds with C (three hydrogen bonds);
• Each pair of nitrogen bases is 0.34 nm away from the following pair and is rotated by 36° with respect to the following pair, meaning that the helix makes a full rotation every 10 pair of bases. The outer diameter is 2 nm long;
• A major groove and a minor groove alternate along the double helix.

Conformations of the Double Helix

The double helix exists in multiple conformations:

• The A form (11 bp/turn) is observed at low humidity and it’s present in certain DNA or protein complex. It’s less hydrated and more compact than the B form. RNA double helix adopts a similar conformation;
• The B form (10 bp/turn) is observed at high humidity and it most closely corresponds to the average structure of DNA under physical conditions;
• The Z form (12 bp/turn) is left-handed and its backbone presents a zig-zag trend.

DNA Replication

DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule and it’s the most essential part for biological inheritance.

According to Watson and Crick’s model of DNA replication, this process is semiconservative, meaning that the two strands of the parental double helix unwind and each strand is a template for a new daughter strand by base-pairing rules. Thus, each daughter molecule is composed of a parental strand and a new complementary one.

Models of DNA Replication

Two other models were proposed in order to explain how the replication works:

• According to the conservative model, the two parental strands act as templates for the synthesis of two new strands and they re-pair. Thus, one of the two DNA molecules obtained is the original molecule, while the other is composed of two new strands;
• According to the dispersive model, the parental molecule is cut into double helix DNA fragments that act as templates for the synthesis of new DNA fragments. These fragments re-pair in order to form a complete double helix DNA, which contains mixed parental and daughter fragments.

In 1958, Meselson and Stahl performed experiments to understand which replication model is correct. They grew E. coli cells on a medium containing the heavy isotope ¹⁵N, which replaced the normal light ¹⁴N isotope and was incorporated in the nitrogen bases inside of the nucleotides. Cells were then grown for many divisions so that the DNA in the cells contained only ¹⁵N. Cells were washed and grown on a medium containing ¹⁴N and samples were taken after one or two cell divisions. DNA was extracted and analyzed on CsCl gradients: the CsCl solution is centrifugated and a density gradient is formed, with the lighter material in the upper part of the test tube and the heavier material in the lower. The DNA present in